Quantification of nucleic acids plays an important part in many molecular biology applications, especially in gene expression analysis. Gene expression analyses are used in particular in the fields of basic research, pharmaceutical research, and molecular diagnostics.
In nucleic acid quantification methods, the concentrations and/or relative or absolute amounts of certain nucleic acids in samples are usually determined. In gene expression analysis, the amounts of mRNA and/or cDNA in biological samples are especially relevant. Northern blot, RNase protection assays, competitive reverse transcription PCR, quantitative reverse transcription PCR (qRT-PCR), microarrays, and high-throughput sequencing methods. Quantitative reverse transcription PCR (qRT-PCR) is most commonly used owing to its high specificity, sensitivity, reproducibility, and speed. However, the results are affected by various critical factors owing to the starting amount and the integrity of the mRNA and owing to the effectiveness of the reverse transcriptase and of the polymerase. In order to ensure comparability of gene expression analyses in different samples, it is necessary to carry out normalization of the amounts determined. Common normalization strategies are based on expression analysis of so-called housekeeping genes. Here, it is assumed that the expression of the housekeeping genes does not differ in different samples. However, this assumption is not correct.
Other normalization methods are based on determining the total RNA in the sample, the sample size (cell number or tissue volume), or on introduced foreign RNA. However, all these approaches have their disadvantages. For example, ribosomal RNA makes up a large part of the total RNA in a cell. Therefore, the amount of total RNA is not always representative of the amount of total mRNA. Normalization methods based on cell number or tissue volume do not take into account that different cells may exhibit different transcriptional activities. Also, these methods do not take into account mRNA quality and the effectiveness of the reverse transcriptase and of the polymerase. Although foreign RNA introduced into the sample takes into account the effectiveness of the enzymes during reverse transcription PCR, it does not take into account the amount and quality of the starting mRNA in the sample.